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Abstract
In vitro plant regeneration addresses basic questions of molecular reprogramming in the absence of embryonic positional cues. The process is highly dependent on the genotype and explant characteristics. However, the regulatory mechanisms operating during organ differentiation from in vitro cultures remain largely unknown. Recently, miRNAs have emerged as key regulators during embryogenic callus induction, plant differentiation, auxin responses and totipotency. Here, we explored how development-related miRNA switches the impact on their target regulation depending on physiological and molecular events taking place during maize Tuxpeño VS-535 in vitro plant regeneration. Three callus types with distinctive regeneration potential were characterized by microscopy and histological preparations. The embryogenic calli (EC) showed higher miRNA levels than non-embryogenic tissues (NEC). An inverse correlation for miR160 and miR166 targets was found during EC callus induction, whereas miR156, miR164 and miR394 displayed similar to their targets RNA accumulation levels. Most miRNA accumulation switches took place early at regenerative spots coincident with shoot apical meristem (SAM) establishment, whereas miR156, miR160 and miR166 increased at further differentiation stages. Our data uncover particular miRNA-mediated regulation operating for maize embryogenic tissues, supporting their regulatory role in early SAM establishment and basipetala growth during the in vitro regeneration process.
Highlights
In vitro plant regeneration is usually achieved through somatic embryogenesis (SE) or de novo shoot organogenesis [1]
We conclude that the initial miRNA and target levels in the callus underlie its regenerative potential and guide its ability to promote shoot appearance during plant regeneration from maize VS-535 in vitro cultures
We found a noteworthy higher accumulation of miRNA targets in embryogenic calli (EC) than in Y-non-embryogenic callus (NEC) and immature embryos (IE) (Figure 4; lower panels), except for SBP23 targeted by miR156, which was more accumulated in IE
Summary
In vitro plant regeneration is usually achieved through somatic embryogenesis (SE) or de novo shoot organogenesis [1]. Somatic embryo induction represents full expression of totipotency, while shoot regeneration reflects cell pluripotency [2]. Both regeneration pathways are dependent on phytohormone perception, cell division and dedifferentiation to acquire organogenic competence, organ initiation and development. Other genotypes cultured in different geographic regions have proven to efficiently undergo SE and plant regeneration. Such is the case of a Chinese inbred elite line, 18-599R [9] and the Mexican improved variety VS-535 developed by Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias (INIFAP) from the Tuxpeño landrace genotype [10,11]. The culture medium, photoperiod, phytohormone ratio and other conditions of the in vitro culture can be adjusted to promote either SE or organogenesis from the induced EC [12]
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